描述该项目由奖号5R01AI116895从过敏和传染病国家研究所的支持。内容完全是作者的责任，并不一定代表国家过敏和传染病研究所和美国国立卫生研究院的官方意见。

1.生成样本<ol><li>热激活C.难辨孢子在65℃下30分钟，并存储在冰上。 注:C.如先前7,9,10所述艰难梭菌芽孢可以产生并纯化。 </li><li>准备在X + 1毫升，其中X是在测定期间要采取的样本数萌发溶液。 注意:萌发溶液是特定细菌的物种孢子正在研究中。用于测定C.难辨孢子萌发，我们使用的10mM Tris（pH值7.5），150毫摩尔NaCl，在去离子水中的100mM甘氨酸和10mM牛磺胆酸的溶液。 </li><li>在开始测定前，画出萌发溶液中的1.0 ml样品无孢子作为皮质片段检测空白。 </li><li>加入孢子的外径〜3.0发芽解决方案，并迅速涡旋600。 注意:如果您在B.萌发研究枯草芽孢杆菌 ，我们使用相同的外径600孢子为C.难辨 。 注:孢子这一数额C.期间行之有效的监测皮质片段发布难辨孢子萌发。孢子在该试验中所使用的量应通过实验为每个细菌或应变来确定。 </li><li>在14000×g下加入孢子和离心机在室温下1分钟后，立即删除在1.2 ml样品。这是在零时间点。 </li><li>传送将1.0ml上清液从离心样品到新管用于随后皮质片段分析。 注意:如果DPA需要监控，取出一个单独的100微升样品在使用基于铽荧光9,18,19的测定溶液中的检测DPA的。 </li><li>冷冻在-80℃下收集的样品。 </li><li>定期重复步骤1.4和1.5，直到所有的时间点完成。 注:由于时间的关系所需的离心和取样时间点少次我们的过程期间的2分钟分开是不可能的，所以我们的采样间隔是一次14分钟每2分钟。 </li><li>至于还原糖的检测和定量阳性对照，包括以下少量的正 -acetylglucosamine（纳摩尔）的:0，12.5，25，50，100，250，500，5000。同时作为皮质样品制备这些样品，并与皮质样品，使得标准曲线是有关由萌芽孢子产生的数据一起运行。 </li><li>所有时间点样品的采集后，冻干样品。 注意:我们冻干我们的样品在2ml螺旋盖试管中。 </li></ol> 2.测量皮质碎片<ol><li>在3当量盐酸的120微升补充有1％的苯酚和0.5％β巯基乙醇悬浮冻干样品。 </li><li>传送再悬浮样本以2毫升螺旋盖试管中。 注意:对于重复性，确保所有的冻干样品重悬于和转让。 </li> &lt;利&gt;孵育再循环水浴将样品在95℃下4小时。 </li><li>孵育后，将样品置于冰上，直到他们都很酷。 </li><li>用120微升3 M氢氧化钠的中和样品。 </li><li>添加80μl的饱和碳酸氢钠溶液和80μl的5％的乙酸酐溶液于每个样品和旋涡。 </li><li>在室温下孵育样品10分钟。 </li><li>转移样本回95℃水浴正好3分钟。 注意:此步骤是时间敏感的和更长的时间能影响下游信号的发展。 </li><li>从水浴中取出样品，并在冰上冷却。 </li><li>添加6.54％K 2 B 4 O 7·4H 2 O 400微升，每管和旋涡。 </li><li>孵育在再循环水浴将样品在95℃EXACTLY 7分钟。 注意:此步骤是时间敏感的和更长的时间能影响下游信号的发展。 </li><li>去掉样品，并在冰上地方5分钟。 </li></ol> 3.显色剂<ol><li>虽然样本上冰（步骤2.12），备显色剂。溶解0.320克对 -dimethylaminobenzaldehyde的; 1.9毫升冰醋酸的（DMAB埃利希氏试剂）。 注:试剂是时间，温度和光敏感，并且不应提前进行。 </li><li>在DMAB完全溶解后，加入100微升10 N HCl和旋涡。 </li><li>加入5毫升冰醋酸和旋涡。 </li></ol> 4.显色反应<ol><li>转移100μl的每个冷却皮质样品到一个新的1.5 ml离心管。 </li><li>添加新鲜制备的色溶液至各样品的700微升。 </li><li>立即将样品转移到37℃水浴中孵育整整20分钟。 </li><li>转移200μl的各样品，以清晰的96孔板中。 </li><li>在量化波长585样本一盘读者。样本应开始以黄色和阳性反应将转向紫色。越皮质目前，较深的紫色。 </li></ol>

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The authors have nothing to disclose.

在刺激时，进行发芽的过程孢子丧失其抗损伤性，而无需大分子合成。当孢子萌发被触发时，孢子释放核心DPA，以换取水2。由于休眠孢子的高DPA含量，孢子核心是在强烈渗透压和专门肽，皮质，有助于防止芯从通过作用，据推测，作为屏障，以扩张2肿胀。 上述的方法使用艾氏试剂（黄色溶液），以检测还原糖作为可测量的移位到紫色色调的存在。期间皮质水解，NAG和NAM糖解放（取决于水解酶的程度，糖类可以解放如二糖或多糖）。由盐酸（步骤2.1）的糖苷键的水解与自由还原端7产生单糖。 neutraliz后用等量的氢氧化钠（步骤2.5）ING酸，包围NAG醇类和NAM单糖是由乙酸酐酰化（步骤2.6）13。接着，未反应的乙酸酐是通过加热样品（步骤2.8）转化为乙酸。由于假设由摩根和埃尔森20，终端醛自发地转化为烯醇式。在碱性条件下（步骤2.10），此烯醇形式可以与相邻的乙酰反应以产生其中用Ehrlich试剂（步骤4.3），以产生紫色15,20反应的恶唑衍生物。 上述这种方法产生的检测皮质碎片一致的，可靠的结果，从萌芽孢子，无论是B.枯草和C.难辨 9,16,17。该试验需要特别注意以确保时间，温度和程序的精度进行该反应。如果没有适当关注细节，很容易在反应条件失败或不实验13之间再现。然而，在实验室环境之间的测定的应用中纳入NAG标准助剂。包括在任何跨实验变异性（ 例如，在反应时间的细微变化，或显色剂的有效性），每一个实验对照组的NAG阳性对照标准。仅使用缓冲液条件和孢子不能水解皮质两者的反应的反应，得到了无颜色变化。 这种方法的最常见的问题是跨实验变异或完全没有信号。为了控制的可变性，孢子制剂必须无任何营养细胞碎片。此细胞碎片将包含肽片段（NAG和NAM），并会导致对萌芽孢子高背景信号。以确保纯孢子制剂（&gt; 99.9％孢子），指的是literatu重新为方法，以在生物体准备纯孢子悬浮液进行研究7,9,21-23。 如果该测定没有产生任何可检测的信号，我们观察到，冻干步骤可以引入可变性。由于与该释放真空的力，我们观察到干燥后的样品的粉末可以吹出的样品管。为了减少/防止这种情况发生，使用一个小的注射器针头戳在样品管的顶部孔。引入孔的小区域将受到干扰（粉末形式现在）降低冻干样品的可能性。最后，如​​在上述方法中指出的那样，几个孵育步骤都非常时间敏感。确保所有定时步骤仔细监测。对于该测定的一个重要考虑因素是，皮质碎片必须从发芽孢子被释放以便被检测到。如果皮层被水解成较大的碎片，这些碎片可能太大是RELE通过芽孢外壳ASED。虽然一个重要的考虑因素的故障排除的实验不产生信号时，我们建议，以确认使用的条件发芽最此处列出在溶液中的孢子和其它故障排除之前考虑皮质片段大小作为一个潜在的问题化验。 不是所有的发芽缓冲器/条件是适合于这种检测。因为该测定法检测减少在发芽过程13，它包含还原糖或即，本身的任何开端，还原糖任何发芽缓冲解放糖的存在（ 例如 ，葡萄糖），将导致在该测定中的强烈信号。如果是这种情况，高压液相色谱（HPLC）可用于检测皮质片段24-26的存在。然而，因为不是所有的实验室都配备这样的仪器，本文描述的方法可以通过使用最多的实验室和提供简单且容易量化的结果。 通过发芽细菌孢子已经在很大程度上被研究芽孢杆菌和少数梭菌属2,8。该方法一般保守的，但是存在差异。重要的是，并不是所有的生物体发芽作为模型孢子形成剂9,27观察用孢子萌发的机制。因为发芽在更加多样的生物变得研究，上述测定可用于这些微生物孢子萌发期间检测皮质片段。通过应用该方法对这些新研究的生物体，研究人员能够确定皮质水解作用的各种蛋白质发芽玩。

芽孢是细菌代谢，使细菌在不利的环境中坚持的休眠形式。在许多孢子形成细菌，孢子形成是通过营养剥夺诱导但此过程可通过改变pH值，暴露于氧或其它应力1进行控制。而在其代谢休眠孢子形式，细菌抵抗紫外线辐射，干燥，更高的温度，和冷冻2。最上的孢子形成过程的知识来自在模式生物的研究， 枯草芽孢杆菌 。在孢子形成过程开始于DNA复制和轴向灯丝3,4的形成。非对称隔膜然后把细胞分成两个大小不等车厢。较大的隔间里，母细胞，吞噬较小的隔间里，forespore。无论是母细胞和forespore协调基因表达成熟的孢子母细胞裂解最终，释放多尔曼吨孢子到周围环境中1。 孢子的结构和组合物在许多细菌物种保守的。孢子芯具有比营养细胞低水含量和有丰富的吡啶二羧酸（DPA）1,2。期间形成孢子，DPA被泵入孢子芯换取水。围绕核心的是一种内在的孢子膜的地方，在多数孢子形成的细菌，其中认识到刺激发芽（germinants）的小分子的受体位于2。刚好位于孢子的内膜外是细胞壁肽聚糖的层。一个专门的肽聚糖层（皮层）围绕细胞壁肽聚糖，并且由许多相同的部件，细胞壁肽聚糖[交替的N-乙酰葡糖胺酶（NAG）和N-乙酰胞壁酸（NAM）]的。但是，在皮质的NAM残基的约50％已被转化为胞壁-δ内酰胺5,6- </s了&gt;。期间孢子萌发，这些胞壁-δ内酰胺残基被孢子皮质裂解酶（SCLEs）的认可，因此允许皮质被降解但不细胞壁（必需完成发芽过程）。周围皮质的外膜和外壳蛋白2几层。 控制发芽的方法对于形成孢子的细菌至关重要。当开端受体各自germinants 2交互萌发开始。在许多孢子形成的细菌，这些germinants是氨基酸，糖，核苷酸，离子或它们的2组合。C.难辨孢子萌发是由某些胆汁酸，[ 例如 ，牛磺胆酸（TA）]和氨基酸（ 例如 ，甘氨酸）7-10的组合引发。虽然有在C之间的差异难辨发芽途径和在所研究的途径其他孢子形成细菌，如B.枯草芽孢杆菌，共同所有的降解孢子皮质以允许营养细胞以从发芽孢子2,8成长的绝对要求。皮质降解可由SCLEs CwlJ / SleB来实现或SLEC（如在枯草芽孢杆菌中发现）（如在许多梭菌找到）。皮质水解降低了细胞壁和孢子的约束。这允许充分芯补液，在重新激活许多必需的细胞代谢2蛋白质的一个必要步骤。 当孢子发芽，从相亮状态到相暗状态，这个过程的休眠孢子的变化可以通过在600光密度（OD）的变化来测量 纳米11。先前的一份报告表明，在很多OD这一变化是由于DPA从12孢子释放。在我们最近的研究中，我们试图比较C的定时难辨孢子萌发和监测DPA和皮质碎片9的释放。在这项研究中是至关重要的监测皮质片段的释放，他们开始由萌芽孢子被释放。 此处所使用的比色测 ​​定法是基于与还原末端检测糖的方法开发Ghuysen 等 13。因为其他人描述协议检测还原糖14或修改这些协议15，关于这一问题的文献可能会造成混淆。在这里，我们详细地一步一步方法还原糖的比色检测从发芽C.解放艰难梭菌芽孢。虽然本研究采用C.艰难梭菌芽孢，从其他孢子形成的细菌孢子的发芽过程中释放的还原糖能够与该协议9,16,17进行检测。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2.0 mL screw cap tube</td>
			<td>USA scientific</td>
			<td>1420-3700</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p20 eppendorf pippet</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100-1250uL pipet tips</td>
			<td>VWR</td>
			<td>89079-486</td>
			<td></td>
		</tr>
		<tr>
			<td>1-200uL pipet tips</td>
			<td>VWR</td>
			<td>89079-458</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl-D-glucosamine</td>
			<td>Sigma</td>
			<td>A3286-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid - 10N</td>
			<td>BDH-Aristar</td>
			<td>BDH3032-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Anhydride</td>
			<td>Alfa Aesar</td>
			<td>L04295</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>BDH</td>
			<td>BDH0280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Tetraborate tetrahydrate</td>
			<td>Alfa Aesar</td>
			<td>39435</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>BDH</td>
			<td>BDH8019-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(Dimethylamino)benzaldehyde</td>
			<td>Sigma</td>
			<td>156477-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Saturated Phenol</td>
			<td>Fisher</td>
			<td>BP1750-400</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Aldrich</td>
			<td>M6250-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax M3</td>
			<td colspan="2">Molecular Devices</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid, Glacial</td>
			<td>BDH</td>
			<td>BDH3098-3.8LP</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated, Circulating Water Bath</td>
			<td>VWR Scientific</td>
			<td>Model 1136</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtest 96</td>
			<td>Falcon</td>
			<td>353072</td>
			<td>96 well clear tissue culture plates</td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>VWR</td>
			<td>89000-506</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Blocks</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Machine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting cortex fragments during bacterial spore germination

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming bacterium, Bacillus subtilis. Germination is triggered by small molecule germinants and occurs without the need for macromolecular synthesis. Though differences exist between the mechanisms of spore germination in species of Bacillus and Clostridium, a common requirement is the hydrolysis of the peptidoglycan-like cortex which allows the spore core to swell and rehydrate. After rehydration, metabolism can begin and this, eventually, leads to outgrowth of a vegetative cell. The detection of hydrolyzed cortex fragments during spore germination can be difficult and the modifications to the previously described assays can be confusing or difficult to reproduce. Thus, based on our recent report using this assay, we detail a step-by-step protocol for the colorimetric detection of cortex fragments during bacterial spore germination.

表1示出了使用NAG标准的典型结果。数据被用于产生标准曲线。 表2示出从发芽缓冲器萌发测定补充有100mM的甘氨酸和10mM TA（发芽促进条件）或100mM的甘氨酸只（非发芽条件）的典型结果。在不存在TA，C的艰难梭菌芽孢不发芽并且在皮质片段在溶液中存在的小的变化。然而，在这两个TA和甘氨酸，C的存在艰难梭菌芽孢发芽并释放皮质片段在溶液中。这被观察为在反应样品的时间的增加的OD 585。该测定过程中释放皮质片段（纳摩尔）的总量，使用类似于图1B的标准曲线来计算。在该试验中，大部分的皮质的水解（或释放）为15分钟后开端曝光，这表明大部分孢子在这个时间帧已发芽（ 图2;与先前出版物7一致）。 显影在彩色试剂的样品后，大多数样品可能不会显示紫色肉眼。 图1示出了从N-乙酰氨基葡糖产生的有代表性的样品。许多样品不显示可见紫色。然而，250纳摩尔，500纳摩尔和5000纳摩尔样品出现从阴性对照组（0纳摩尔）目视不同。当在OD 585处测量，所产生的信号最适合的线性回归（R 2 = 0.999）。 <img alt="图1" src="/files/ftp_upload/54146/54146fig1.jpg" /> 图 1: 检测N-乙酰葡糖胺 （A）的N-乙酰氨基葡糖的样品（0纳摩尔，12.5纳摩尔，25纳摩尔，50纳摩尔，100纳摩尔，250纳摩尔，500纳摩尔，5000纳摩尔）按照所述步骤冻干并反应。 （B）的样品加入到96孔板中并在585处检测到的反应材料的量。该信号被用来产生标准曲线。 <img alt="图2" src="/files/ftp_upload/54146/54146fig2.jpg" /> 图 2: 萌发结果的图形表示在表2中的计算纳摩尔皮质与时间的关系。 （●）100毫米甘氨酸+ 10毫米TA; （■）100毫米甘氨酸。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54146/54146fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> NAG浓度（纳摩尔） </td><td> OD 585 </td></tr><tr><td> 0 </td><td> 0.047 </td></tr><tr><td> 12.5</td><td> 0.054 </td></tr><tr><td> 25 </td><td> 0.066 </td></tr><tr><td> 50 </td><td> 0.084 </td></tr><tr><td> 100 </td><td> 0.128 </td></tr><tr><td> 250 </td><td> 0.285 </td></tr><tr><td> 500 </td><td> 0.556 </td></tr><tr><td> 5000 </td><td> 4 </td></tr><tr><td>空白= </td><td> 0.046 </td></tr></tbody></table> 表1:NAG标准。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td colspan="4"> 艰难梭菌孢子萌发</td></tr><tr><td></td><td colspan="4">发芽溶液（10mM的Tris（pH值7.5），150mM的NaCl）中</td></tr><tr><td></td><td colspan="2"> 100毫米甘氨酸+ 10毫米TA </td><td colspan="2"> 100毫米甘氨酸</td></tr><tr><td>时间（分钟） </td><td> OD 585 </td><td>计算（纳摩尔） </td><td> OD 585 </td><td>计算（纳摩尔） </td></tr><tr><td> 0 </td><td> 0.046 </td><td> 0.00 </td><td> 0.047 </td><td> 0.00 </td></tr><tr><td> 2 </td><td> 0.048 </td><td> 2.54 </td><td> 0.048 </td><td> 1.27 </td></tr><tr><td> 4 </td><td> 0.05 </td><td> 5.07 </td><td> 0.049 </td><td> 2.54 </td></tr><tr><td> 6 </td><td> 0.055 </td><td> 11.41 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 8 </td><td> 0.059 </td><td> 16.49 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 10 </td><td> 0.06 </td><td> 17.76 </td><td> 0.051 </td><td> 5.07 </td></tr><tr><td> 12 </td><td> 0.061 </td><td> 19.02 </td><td> 0.05 </td><td> 3.80 </td></tr><tr><td> 14 </td><td> 0.063 </td><td> 21.56 </td><td> 0.051 </td><td> 5.07 </td></tr></tbody></table> 表2:样品孢子Germinati对结果的。

Watch this Scientific Journal Video about Detecting Cortex Fragments During Bacterial Spore Germination at JoVE.com

Detecting Cortex Fragments During Bacterial Spore Germination

Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

检测皮质片段在细菌孢子萌发

The process of endospore germination in Clostridium difficile, and other Clostridia, increasingly is being found to differ from the model spore-forming ...

detecting-cortex-fragments-during-bacterial-spore-germination

Nanyang Technological University

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JoVE Journal

Immunology and Infection

9.3K 次观看.  Texas A&M University. Herein, we describe a colorimetric assay to detect the presence of reducing sugars during bacterial spore germination.

视频: Detecting Cortex Fragments During Bacterial Spore Germination

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

宣传和检测Maedi visna病毒传染性表达绿色荧光蛋白的分子克隆

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

科研

免疫与感染

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Detecting Glycogen in Peripheral Blood Mononuclear Cells with Periodic Acid Schiff Staining

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. Polysaccharides such as glycogen, glycoproteins, and glycolipids stain bright magenta making it easy to enumerate positive and negative cells within the tissue. In muscle cells PAS staining is used to determine the glycogen content in different types of muscle cells, while in blood cell samples PAS staining has been explored as a diagnostic tool for a variety of conditions. Blood contains a proportion of white blood cells that belong to the immune system. The notion that cells of the immune system possess glycogen and use it as an energy source has not been widely explored. Here, we describe an adapted version of the PAS staining protocol that can be applied on peripheral blood mononuclear immune cells from human venous blood. Small cells with PAS-positive granules and larger cells with diffuse PAS staining were observed. Treatment of samples with amylase abrogates these patterns confirming the specificity of the stain. An alternate technique based on enzymatic digestion confirmed the presence and amount of glycogen in the samples. This protocol is useful for hematologists or immunologists studying polysaccharide content in blood-derived lymphocytes.

Periodic acid Schiff staining is a technique that visualizes the polysaccharide content of tissues. This article demonstrates periodic acid Schiff staining protocol adapted for use on peripheral blood mononuclear cells purified from human venous blood. Such samples are enriched for lymphocytes and other white blood cells of the immune system.

与碘酸雪夫染色检测糖原的外周血单个核细胞

Periodic acid Schiff (PAS) staining is an immunohistochemical technique used on muscle biopsies and as a diagnostic tool for blood samples. ...

Electroporation of Functional Bacterial Effectors into Mammalian Cells

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high-throughput characterization of pathogen proteins in host cells including subcellular targeting and function of virulence proteins.

Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

电功能性细菌效应器到哺乳动物细胞

The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Evaluation of Extracellular Vesicle Function During Malaria Infection

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible for most malaria-related deaths globally. Several factors including parasite sequestration in tissues, vascular dysfunction, and inflammatory responses influence the evolution of the disease in malaria-infected people. P. falciparum-infected red blood cells (iRBCs) release small extracellular vesicles (EVs) containing different kinds of cargo molecules that mediate pathogenesis and cellular communication between parasites and host. EVs are efficiently taken up by cells in which they modulate their function. Here we discuss strategies to address the role of EVs in parasite-host interactions. First, we describe a straightforward method for labeling and tracking EV internalization by endothelial cells, using a green cell linker dye. Second, we report a simple way to measure permeability across an endothelial cell monolayer by using a fluorescently labeled dextran. Finally, we show how to investigate the role of small non-coding RNA molecules in endothelial cell function.

In this work, we describe protocols to investigate the role of extracellular vesicles (EVs) released by Plasmodium falciparum infected erythrocytes. In particular, we focus on the interactions of EVs with endothelial cells.

疟疾感染中胞外囊泡功能的评价

Malaria is a life-threatening disease caused by Plasmodium parasites, with P. falciparum being the most prevalent on the African continent and responsible ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

铜绿假单胞菌感染肺内皮细胞后细胞毒性 Amyloids 的检测方法

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Spore Adsorption as a Nonrecombinant Display System for Enzymes and Antigens

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure makes the spore extremely stable and resistant and has suggested the use of the spore as a platform to display heterologous molecules. So far, a variety of antigens and enzymes have been displayed on spores of Bacillus subtilis and of a few other species, initially by a recombinant approach and, then, by a simple and efficient nonrecombinant method. The nonrecombinant display system is based on the direct adsorption of heterologous molecules on the spore surface, avoiding the construction of recombinant strains and the release of genetically modified bacteria in the environment. Adsorbed molecules are stabilized and protected by the interaction with spores, which limits the rapid degradation of antigens and the loss of enzyme activity at unfavorable conditions. Once utilized, spore-adsorbed enzymes can be collected easily with a minimal reduction of activity and reused for additional reaction rounds. In this paper is shown how to adsorb model molecules to purified spores of B. subtilis, how to evaluate the efficiency of adsorption, and how to collect used spores to recycle them for new reactions.

This protocol focuses on the use of bacterial spores as a &#34;live&#34; nanobiotechnological tool to adsorb heterologous molecules with various biological activities. The methods to measure the efficiency of adsorption are also shown.

作为酶和抗原的非重组显示系统的孢子吸附

The bacterial spore is a metabolically quiescent cell, formed by a series of protective layers surrounding a dehydrated cytoplasm. This peculiar structure ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...